Mitochondrial diseases are devastating disorders for which there is no cure and no proven treatment. About 1 in 2000 individuals are at risk of developing a mitochondrial disease sometime in their lifetime. Half of those affected are children who show symptoms before age 5 and approximately 80% of these will die before age 20. The human suffering imposed by mitochondrial and metabolic diseases is enormous, yet much work is needed to understand the genetic and environmental causes of these diseases. Mitochondrial genetic diseases are characterized by alterations in the mitochondrial genome, as point mutations, deletions, rearrangements, or depletion of the mitochondrial DNA (mtDNA). The mutation rate of the mitochondrial genome is 10-20 times greater than of nuclear DNA, and mtDNA is more prone to oxidative damage than is nuclear DNA. Mutations in human mtDNA cause premature aging, severe neuromuscular pathologies and maternally inherited metabolic diseases, and influence apoptosis. The primary goal of this project is to understand the contribution of the replication apparatus in the production and prevention of mutations in mtDNA. Since the genetic stability of mitochondrial DNA depends on the accuracy of DNA polymerase gamma (pol gamma), we have focused this project on understanding the role of the human pol gamma in mtDNA mutagenesis. Human mitochondrial DNA is replicated by the two-subunit gamma, composed of a 140 kDa subunit containing catalytic activity and a 55 kDa accessory subunit. The catalytic subunit contains DNA polymerase activity, 3'-5' exonuclease proofreading activity, and 5'dRP lyase activity required for base excision repair. As the only DNA polymerase in animal cell mitochondria, pol gamma participates in DNA replication and DNA repair. The 140 kDa catalytic subunit for pol gamma is encoded by the nuclear POLG gene. To date nearly 200 pathogenic mutations in POLG that cause a wide spectrum of disease including Progressive external ophthalmoplegia (PEO), parkinsonism, premature menopause, Alpers syndrome, mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) or sensory ataxic neuropathy, dysarthria, and ophthalmoparesis (SANDO). Human mitochondrial DNA polymerase gamma (polg) is solely responsible for the replication and repair of the mitochondrial genome. Thus far, predicting the severity of mitochondrial disease based the magnitude of deficiency in polg activity has been difficult. In order to understand the relationship between disease severity in patients and enzymatic defects in vitro, we characterized the molecular mechanisms of four polg mutations, A957P, A957S, R1096C and R1096H, which have been found in patients suffering from aggressive Alpers syndrome to mild progressive external ophthalmoplegia. The A957P mutant showed the most striking deficiencies in the incorporation efficiency of a correct deoxyribonucleotide triphosphate (dNTP) relative to wild-type polg, with less, but still significant incorporation efficiency defects seen in R1096H and R1096C, and only a small decrease in incorporation efficiency observed for A957S. Importantly, this trend matches the disease severity observed in patients very well (approximated as A957P > R1096C > R1096H > A957S, from most severe disease to least severe). Further, the A957P mutation conferred a two orders of magnitude loss of fidelity relative to wild-type polg, indicating that a buildup of mitochondrial genomic mutations may contribute to the death in infancy seen with these patients. We conclude that characterizing the unique molecular mechanisms of polg deficiency for physiologically important mutant enzymes is important for understanding mitochondrial disease and for predicting disease severity. Mammalian mitochondrial DNA (mtDNA) is replicated by the heterotrimeric polg comprised of a single catalytic subunit, encoded by POLG, and a homodimeric accessory subunit encoded by the POLG2 gene. While the catalytic subunit has been shown to be essential for embryo development, genetic data regarding the accessory subunit are lacking in mammalian systems. We generated a heterozygous (Polg2(+/-)) and homozygous (Polg2(-/-)) knockout (KO) mice. Polg2(+/-) mice are haplosufficient and develop normally with no discernable difference in mitochondrial function through 2 years of age. In contrast, the Polg2(-/-) is embryonic lethal at day 8.0-8.5 p.c. with concomitant loss of mtDNA and mtDNA gene products. Electron microscopy shows severe ultra-structural defects and loss of organized cristae in mitochondria of the Polg2(-/-) embryos as well as an increase in lipid accumulation compared with both wild-type (WT) and Polg2(+/-) embryos. Our data indicate that Polg2 function is critical to mammalian embryogenesis and mtDNA replication, and that a single copy of Polg2 is sufficient to sustain life. Acrolein, a mutagenic aldehyde, is produced endogenously by lipid peroxidation and exogenously by combustion of organic materials, including tobacco products. Acrolein reacts with DNA bases forming exocyclic DNA adducts, such as g-hydroxy-1,N2-propano-2'-deoxyguanosine (g-HOPdG) and g-hydroxy-1,N6-propano-2'-deoxyadenosine (g-HOPdA). The bulky g-HOPdG adduct blocks DNA synthesis by replicative polymerases but can be bypassed by translesion synthesis polymerases in the nucleus. Although acrolein-induced adducts are likely to be formed and persist in mitochondrial DNA, animal cell mitochondria lack specialized TLS polymerases to tolerate these lesions. Thus, it is important to understand how polg, the sole mitochondrial DNA polymerase in human cells, acts on acrolein-adducted DNA. To address this question, we investigated the ability of polg to bypass the minor-groove &#947;-HOPdG and major-groove g-HOPdA adducts using single nucleotide incorporation and primer extension analyses. The efficiency of polg-catalyzed bypass of g-HOPdG was low and surprisingly, polg preferred to incorporate purine nucleotides opposite the adduct. Polg also exhibited 2-fold lower rates of excision of the misincorporated purine nucleotides opposite g-HOPdG compared to the corresponding nucleotides opposite dG. Extension of primers from the termini opposite g-HOPdG was accomplished only following error-prone purine nucleotide incorporation. However, polg preferentially incorporated dT opposite the g-HOPdA adduct and efficiently extended primers from the correctly paired terminus, indicating that g-HOPdA is probably non-mutagenic. In summary, our data suggest that acrolein-induced exocyclic DNA lesions can be bypassed by mitochondrial DNA polymerase but in the case of the minor-groove &#947;-HOPdG adduct, at the cost of unprecedentedly high mutation rates. Deletions, mostly between the 13-nucleotide direct repeats, accumulate in mtDNA in humans during aging and Kearns-Sayre syndrome. The importance of mitochondrial DNA (mtDNA) deletions in the progeroid phenotype of exonuclease-deficient POLG mice has been intensely debated. To test whether exonuclease activity attenuates deletions between 21 nucleotide direct repeats, heteroallelic yeast strains with a wild-type MIP1 chromosomal allele and an exonuclease deficient mip1 allele expressed from a centromeric plasmid were assayed. Mitochondrial genomes from these strains contain an ARG8 insertion, flanked by direct repeats, in the mitochondrial6 encoded COX2 gene. Deletion between direct repeats simultaneously results in loss of ARG8 and restoration of COX2, allowing for growth on glycerol-containing media. We showed that disruption of Mip1 exonuclease activity increases mtDNA deletions 160-fold, whereas disease-associated polymerase variants were mostly unaffected, suggesting that exonuclease activity is vital to avoid deletions during mtDNA replication. However, homologous POLG disease mutations in MIP1 did not increase the deletion formation.